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Background Non-muscle-invasive bladder cancer (NMIBC) remains a therapeutic challenge, especially in BCG-unresponsive cases. Recombinant non-replicative modified vaccinia Ankara (rMVA) vectors offer a promising immunotherapy platform. Our first-generation virus, rMVA/MQ710 (MVA∆E5R-Flt3L-OX40L), deletes the viral immune evasion gene E5R and expresses membrane-anchored Flt3L and OX40L to activate innate and adaptive immunity via cGAS/STING and type I IFN signaling. The human version of MQ710 is currently in a Phase I clinical trial for solid tumors at Memorial Sloan Kettering Cancer Center. Here, we assessed intravesical delivery of second-generation rMVA/MQ833, encoding IL-12 tethered to the extracellular matrix and with two additional deletions of viral immune evasion genes, E3L and WR199, in an orthotopic murine bladder cancer model.Methods C57BL/6J mice were implanted intravesically with 2.5 × 10 5 MB49 bladder tumor cells and treated with MQ710, MQ833, or PBS (1 x 108 pfu in 75 µL intravesically, every 72 hours for up to 60 days). Tumor growth and survival were monitored. Mice achieving complete response were rechallenged subcutaneously with ten times tumor dose; naïve mice served as controls. To assess abscopal effects, mice were co-implanted with orthotopic and subcutaneous tumors, and only the bladder tumors received MQ833. For mechanistic studies, CD45+ cells from MQ710, MQ833-treated bladder and untreated cutaneous tumors were subjected to integrated single-cell RNA and TCR sequencing. Tumors treated with PBS were used as controls.Results Intravesical rMVA treatment resulted in 33% (MQ710) and 55% (MQ833) cure rates, with durable protection upon rechallenge. In mice with dual tumor sites, MQ833 treatment of bladder tumors alone led to regression of distant subcutaneous tumors, demonstrating robust abscopal effects. Integrated scRNA-seq and TCR-seq revealed distinct immune landscapes in bladder vs. subcutaneous tumors. MQ710 and MQ833 induced clonal expansion of effector CD8 +T cells across both sites, with enrichment of cytotoxic and interferon-stimulated genes. Notably, virotherapy promoted recruitment of Nos2+ and ISG+ neutrophils in both tumor sites, with stronger effects observed with MQ833.Conclusions Intravesical delivery of rMVA vectors MQ710 and MQ833 elicits potent local and systemic antitumor responses, including memory formation, T cell clonal expansion, and neutrophil-mediated immunity. These findings support the clinical development of rMVA-based intravesical immunotherapy for NMIBC.Acknowledgements This work was supported by the Center for Experimental Therapeutics Grant (L.D.), Rockefeller University Center for Clinical and Translational Science (RUCCTS) (L.D, J.A.L, and J.C.O). J.A.L is a Clinical Scholar at the Rockefeller University.